The internal urethral sphincter, which is the smooth muscle of the bladder neck, controls the complex micturition system by relaxing during the voiding phase and contracting during the storage phase. Relaxation impairment of this muscle causes a voiding dysfunction. In severe cases, patients need an intermittent urethral catheterization for voiding, which reduces their quality of life and poses a risk of infection.
Nitric oxide (NO) plays an important role in the relaxation of the internal urethral sphincter; however, NO has not been used for voiding dysfunction for two reasons. First, it is difficult to avoid systemic side effects, such as headache or hypotension. Second, the internal urethral sphincter must be relaxed only during the voiding phase. Therefore, we developed a novel light-reactive NO donor, “NORD-1”. It is possible to regulate the NO release temporally and spatially by irradiating NORD-1 with red light. In this study, we examined whether it was possible to control the relaxation of the bladder neck of intact rats by using NORD-1 and red light.

We used 10-11-week-old male and female Wistar/ST rats. In the NORD-1 group, 10^-4 M NORD-1 (500 µL) was instilled into the bladder via a PE-50 tube inserted into the bladder dome under anesthesia. In the vehicle group, we administered the vehicle. The rats were kept in the supine position for 20 min, following which, the bladder was harvested. Next, the bladder was separated into a circular bladder neck specimen and a longitudinal bladder body strip for isometric tension study. Carbachol (10^-5 M) was added to the organ bath to induce precontraction. After equilibration, the specimens were irradiated with red light (8 mW/cm2, 30 mW/cm2, 58 mW/cm2, and 121 mW/cm2) and their relaxation responses were measured. We also evaluated their response in the presence of 10^-5 M [1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ), an inhibitor of the soluble guanylyl cyclase (sGC). All measurements were performed with 10^-5 M NG-nitroarginine methyl (L-NAME), an inhibitor of the endogenous NO synthase. Moreover, bladder necks were harvested from rats treated with vehicle or NORD-1 and frozen sections were prepared to analyze the permeability of NORD-1. Paired samples t-test with the Bonferroni correction was used for the statistical analysis of the difference in response to each light intensity, and ANOVA and the Bonferroni-type multiple t-tests were used for the analysis of the response with or without NORD-1 and ODQ.

Fig. 1A shows representative charts of the female rat’s bladder neck response to irradiation with red light under each condition. In the vehicle group, the bladder neck specimens did not respond to irradiation and were relaxed due to the application of sodium nitroprusside, an NO donor (Fig. 1A). In contrast, in the NORD-1 group, the bladder neck specimens were relaxed when irradiated, and the response was significantly increased in proportion to the light intensity (Fig. 1A, 1B). In addition, the tension returned as soon as the light was turned off. These responses were significantly inhibited in the presence of ODQ (Fig. 1A, 1C). Contrarily, the bladder body specimens of both groups did not relax when irradiated. There were no differences based on sex in the relaxation responses. According to the histological evaluation, NORD-1 was localized in the urothelium, but not in the smooth muscle of the bladder neck specimens (Fig. 2).

We succeeded in controlling the start and end of the relaxation of bladder neck specimens obtained from intact rats by using NORD-1 and light irradiation. In contrast, the bladder body specimens did not react to them. These differences may originate from a cellular expression of the sGC. The sGC is an intracellular receptor of NO, which induces the relaxation of smooth muscles. It was reported that sGC was not detected in the smooth muscle cells of the bladder body, but was present in those of the bladder neck [1]. Thus, the bladder neck relaxed via the NO/sGC signal when NORD-1 was applied and irradiated, while the bladder bodies did not respond. However, NORD-1 is localized in the urothelium rather than the smooth muscle of the bladder neck. It is known that NO migrates from the producer cells to the target cells freely and quickly. In this study, NO was produced in the urothelial cells of the bladder neck specimens and transferred to the smooth muscle cells immediately.

This study is the first to apply a light-reactive NO releaser to a bladder neck specimen. This method may resolve the problems that the usual NO donors face by controlling an NO release spatially and temporally. Light-reactive NO releaser and light irradiation may be a novel therapy for voiding dysfunction that affects the patient physically and mentally.

Lies B, Groneberg D, Friebe A. Correlation of cellular expression with function of NO-sensitive guanylyl cyclase in the murine lower urinary tract. J Physiol. 2013 Nov 1;591(21):5365-75.